It is increasingly desirable to provide a rapid high sensitivity system to detect low levels of ligands in body fluids, plant extracts, environmental samples, tissue samples and enrichment broths. Ideally, such systems should have a minimal number of procedural steps and yield reliable results, even when used by untrained persons. A ligand may be a specific sequence of amino acids or molecule found on proteins such as an antibody, protein receptors, bacterial/microbial peptides, hormone, or drug that binds to a receptor. A receptor is any of various specific protein molecules in surface membranes of cells and organelles to which complementary molecules, such as hormones, neurotransmitters, antigens, or antibodies, may become bound. The ligand may also be a chemical intermediate or reactant with an analyte. Analytes are substances that bind to a ligand. Ligands or analytes may also be peptides, drugs, carbohydrates, haptens, chemicals, chemical reaction with an intermediate compound, and the like.
To a significant extent, many known tests presently available for detecting ligands are either time consuming, labor intensive, or in need of instrumental assistance to read results. Most known tests also lack an acceptable degree of sensitivity or specificity. This is unfortunate since rapid testing is important for diagnosis and treatment of various physiological conditions; detection of certain strains of micro-organisms; determination of the most appropriate antibiotic treatment of a patient; detection of drug analytes in individuals; detection of cancer cells in a patient's bio-fluid; detection of antibody to a microbial agent; detection of a disease-state protein; and the like.
Although known types of ligand-receptor assays have been used to detect the presence of various substances, such as ligands, there is a need in the art to provide a rapid, high sensitivity assay requiring a minimum degree of skill from a user. Rapid test assay devices for field use, such as in a home or doctor's office are known in the art for detecting proteins, peptides, drugs, carbohydrates, haptens, chemicals, chemical reaction with intermediate compounds, and the like. Such devices are referred to as one-step lateral flow or one-step immuno-chromatographic assays. These types of assay devices require a minimal number of steps and can be performed by an untrained person. Examples of these test devices abound, and the individual components of a typical one-step lateral flow test are described below:
A first type of test device involves a test strip of rectangular or square dimensions made of a vinyl, polypropylene, or other pliable or non-pliable plastic laminate to serve as a backing to hold in place other test components that are on an adhesive bond on the backing. A ligand, protein or analyte binding membrane having discreet zones of immuno-reactive proteins or substances immobilized or attached, are usually in linear impregnation or spotted through a dropper onto the membrane. The membrane may be composed of nylon, nitrocellulose, mixed cellulose esters, polysulfones, and the like. If the assay is used to detect an animal antibody, the immuno-reactive protein coated on the membrane may be a ligand to which antibody contained in a positive sample reacts. These immuno-reactive proteins used to coat the membrane are typically native or recombinant proteins derived from Human Immunodeficiency Virus (HIV), human T-cell lymphotropic viruses (HTLV), Mycobacterium tuberculosis (TB), and the like. In a second construct, the membrane may also be impregnated with anti-antibody to capture total class and subclass immunoglobulins. In this case, the specific antibody in the sample may react with native or recombinant ligand proteins, such as HIV, HTLV, or TB, coated onto the surface of the conjugate particles. A third construct may have native or recombinant protein antigens coated on both conjugate and membrane surfaces that react with separate binding domains on the antibody. If the membrane is detecting antigen as the analyte, the surface may be impregnated with antibody or ligand reactive with the antigen. Constructs one, two and three are known as “sandwich” type rapid assays, since the analyte being detected is captured (sandwiched in between) by both the immuno-conjugate and the membrane surface. A fourth method is a competitive inhibition assay, wherein sample analyte at detectable levels either saturates the anti-analyte conjugate or saturates the immobilized anti-analyte capture line on the membrane resulting in no visible line formation. A negative reaction in this case results in a visible line formation, due to the binding of the conjugate with the test line.
A second group of test devices involve a fibrous membrane, such as, for example, glass, polyester, cotton, or spun polyethylene, in contact with a membrane containing ligand and bound to densely colored particles such as latex, gold, silver, selenium, carbon, and the like. The bound ligand is complementary to the assay being constructed and reacts with the analyte being detected. The coated colored particles are often described as an immuno-conjugate. Sufficient molecules of ligand are coated onto the surface of the colored particles so that when a positive reaction does occur the discreet, striped, or spotted zones on the membrane surface are visible to the naked eye. A sample negative for the ligand being detected may leave a white zone in a sandwich type immuno-assay. If the assay is a competitive inhibition type, a negative sample yields a visible line or spot. The colored particles may be dried down onto the fibrous pad or membrane and placed at the dorsal end (at the opposite end of the absorbent pad or membrane) of the membrane. Release agents may be contained in the dried down colored particles to facilitate re-hydration of the particles, allowing them to react with the analyte being detected.
A third group of test devices include a fibrous sample receiving pad or membrane, such as glass, polyester, cotton, or spun polyethylene, that is partially in contact with the immuno-conjugate and serves as a reservoir for absorbing and releasing sample. The sample may contain chemicals to facilitate reactive qualities of the assay. The sample may be any biological fluid (bio-fluid) such as tissue extracts, blood, serum, plasma, tears, perspiration, urine, or saliva. The sample may also be derived from an environmental extract, plant extract, or microbial enrichment broth. When a sample or diluted sample is applied to the sample receiving pad or membrane, the movement of liquid is chromatographic and unidirectional towards the absorbent pad or membrane. During migration, the sample re-hydrates the colored particles and reacts with ligand bound to the particles.
Other test devices are known. For example, one has a bulbous absorbent pad or membrane, which serves as a reservoir for absorbing all liquid components that pass through the membrane. The absorbent material is typically cotton or paper. Another has a zone applied to the membrane containing a control line indicating sample was applied, or that the assay's immuno-conjugate is functional. The control can react with the sample or conjugate. Still another has buffered diluent often used to dilute and condition the sample being detected, allowing for exotic chemistries to occur, thus improving assay performance. The diluent is composed of salt solutions, detergents, and the like and may be applied from a dropper tip, vials, or is contained in a sample vial. And finally another has a plastic or cardboard housing to contain, provide support, and allow ease of use for the whole strip.
It is important to note that in the known prior art the conjugate pad or membrane is in the same plane as the membrane, and overlaps, or is in constant contact with the membrane. Further, the conjugate is in dry form disposed or deposited in the flow path upstream of the test site. This gives rise to a very specific flow path wherein the sample re-hydrates the dried conjugate and travels through the absorbent pad or membrane. The flow path is known as an absorbent flow path since the liquid sample is absorbed by the sample receiving pad or membrane, and next wets the conjugate pad or membrane. Typically, the amount of sample required to wet the conjugate pad or membrane is about 10 to 20 microliters (μl). The sample and conjugate mixture subsequently travels through the conjugate pad or membrane, then contacts the membrane and flows onto the test (flow) membrane.
In use, in most lateral flow assays using antigen-antibody reactions, the immuno conjugate is composed of synthetic conjugates having latex microparticles, enzymatic, fluorescent, or visually observable metal sol tags. In all these assays, there is a receptor, for example an antibody, which is specific for the selected ligand or antigen, a means for detecting the presence, and often a quantity of the ligand-receptor reaction product. Examples of such qualitative assays include blood typing and most types of urinalysis. For these semi-quantitative type positive/negative tests, visually observable indicia such, as the presence of agglutination or a color change are preferred.
Positive/negative assays (or semi-quantitative) must still be very sensitive because of the often small concentrations of the ligands of interest in the test fluid. False positives may be troublesome, particularly with agglutination and other rapid detection methods such as dipstick and color change tests. Because of these problems, sandwich and competitive inhibition assays and other detection methods that use metal sols or other types of colored particles have been developed in an attempt to increase sensitivity of detection. In addition, the use of direct labels attached to one of the specific binding ligands produces instant analytical results without the need to add further reagents. Nevertheless, these techniques have not solved all problems related to sensitivity, neither have they been reliable in obtaining results with a minimal number of method steps encountered in these rapid detection methods.
Various other techniques have been devised for labeling one member of a specific binding pair so that the binding reaction may be indirectly observed. The results of specific binding reactions are generally not directly observable. Useful labels include radiolabels, chromophores, fluorophores, (the presence of which may be detected by means of radiation detectors), spectrophotometers, the naked eye, and the like. Where members of a specific binding pair are tagged with an enzyme label, their presence may be detected by the enzymatic activation of a reaction system including a signal generating a substrate/cofactor group wherein a compound, such as a dye, is activated to produce a detectable signal.
Other specific binding assay devices are known in the art having vertically arranged elements including the following: a porous capture material impregnated at a reaction site with a member of a specific binding pair such as an antibody or an antigen; a removable prefilter disposed above the capture material; and, a blotter disposed below the capture material. A sample liquid such as blood, serum or other bio-fluid is added to the device wherein the prefilter removes particulates and other impurities from the sample. These impurities would otherwise be trapped on top of the specific binding capture material. Analyte substances within the sample are trapped by means of specific binding reactions with their specific binding partners on the capture material. Non-analyte components of the sample solution pass through the capture material and are absorbed by the blotter. Wash steps may be carried out to remove non-analyte components from the capture material and additional reagents such as enzyme substrates, cofactors and dye precursors may be added to the capture material to indicate the presence or absence of analyte at the reaction site. The prefilter should then be removed so that the presence or absence of analyte at the reaction site may be visually determined. Unfortunately, while these devices are somewhat useful, they suffer from limitations in capture efficiency and sensitivity because most of the analyte in the sample 10 material flows around, rather than through, the reaction site on the capture material.
Several one-step lateral flow immunoassay devices, having a strip capable of transporting a developing liquid by capillary action having a first zone for receiving a sample, a second zone impregnated with a first reagent capable of being transported by the developing liquid, and a third zone impregnated with a second reagent, are known in the art (See generally, U.S. Pat. Nos. 4,366,241; 4,094,647; 4,235,601; 4,361,537; and 6,841,159). U.S. Pat. Nos. 6,352,862; 5,622,871; and 5,120,643 also relate to one-step lateral flow tests wherein the conjugate is in dry form along the flow path.
A major disadvantage of all of the known prior art is that the sample rehydrates the dried conjugate along the flow path. Thus, the majority of sample applied is not involved in the immunological reaction. For example, between 10 μl to 20 μl of sample is all that is required to fully rehydrate a 5 by 5 mm gold pad or membrane with the conjugate pad or membrane composed of polyester, glass, nylon, cellulose or other fibers. Since the conjugate pad or membrane is in the device flow path and in contact with the membrane, a minimal remaining sample is involved in the immunological reaction. The remaining 50 μl to 150 μl of sample acts as a liquid front by pushing the reacted analyte conjugate complexes through the flow path. Thus, a severe limitation of the one-step lateral flow assay is the limited amount of sample actually involved in the reaction with the conjugate. None of the known prior art teach or suggest locating the conjugate pad or membrane on the reverse side of the strip, and not in contact with either the sample receiving pad or the membrane. By locating the conjugate pad or membrane on the reverse side of the membrane, the entire sample first rehydrates the conjugate resulting in a uniform dispersion of conjugate through the entire applied sample. Further, in a one-step lateral flow assay, as a sample mixes with a dry conjugate, incomplete mixing often yields conjugate particles with vastly different numbers of captured analyte molecules. This alone may be a source of decreased sensitivity since the maximum number of analyte molecules are not captured on the surface of the conjugate particles.
Other designs are known in the art also include two-step assays. These often require the conjugate pad or membrane to be physically and spacially removed from the test strip. For example, there are assays known in the art where the conjugate is contained in book form, or a “male to female” molded apparatus. In the book form, the test strip is located on one panel of the book while the conjugate is located on the other panel. The test requires the user to close both halves for the test to begin. With the “male to female” apparatus, the sample is applied to the male portion of the apparatus, and the two halves are closed to initiate the reaction. Another format has the conjugate dried and located on a separate sampling stick. The sampling stick is mixed with a sample and is then fixed in the cassette sample well. For example, some two step assay platforms are known in the art, such as indicated in U.S. Pat. No. 6,824,997 and U.S. Pat. No. 5,418,171. These tests have a significant drawback though. The end user must perform an additional step to physically bring the membrane and conjugate into contact after the sample has been adequately mixed.
Thus, there is a desire and need in the art for a high sensitivity assay system and apparatus to rapidly detect low levels of ligands in a small sample size of fluid. Such new tests should involve a minimal number of procedural steps while at the same time, yielding reliable results, even when used by untrained persons. Such a system and apparatus would be useful in areas of infectious disease, pregnancy, microbial detection, ovulation, cancer marker identification, autoimmunity, cardiac markers, biowarfare agents, allergy, drugs of abuse and environmental monitoring, and the like.